CSN1S1, CSN3 and LPL: Three Validated Gene Polymorphisms Useful for More Sustainable Dairy Production in the Mediterranean River Buffalo.

The search for DNA polymorphisms useful for the genetic improvement of dairy farm animals has spanned more than 40 years, yielding relevant findings in cattle for milk traits, where the best combination of alleles for dairy processing has been found in casein genes and in DGAT1. Nowadays, similar results have not yet been reached in river buffaloes, despite the availability of advanced genomic technologies and accurate phenotype records. The aim of the present study was to investigate and validate the effect of four single nucleotide polymorphisms (SNP) in the CSN1S1, CSN3, SCD and LPL genes on seven milk traits in a larger buffalo population. These SNPs have previously been reported to be associated with, or affect, dairy traits in smaller populations often belonging to one farm. A total of 800 buffaloes were genotyped. The following traits were individually recorded, monthly, throughout each whole lactation period from 2010 to 2021: daily milk yield (dMY, kg), protein yield (dPY, kg) and fat yield (dFY, kg), fat and protein contents (dFP, % and dPP, %), somatic cell count (SCC, 103 cell/mL) and urea (mg/dL). A total of 15,742 individual milk test day records (2496 lactations) were available for 680 buffalo cows, with 3.6 ± 1.7 parities (from 1 to 13) and an average of 6.1 ± 1.2 test day records per lactation. Three out four SNPs in the CSN1S1, CSN3 and LPL genes were associated with at least one of analyzed traits. In particular, the CSN1S1 (AJ005430:c.578C>T) gave favorable associations with all yield traits (dMY, p = 0.022; dPY, p = 0.014; dFY, p = 0.029) and somatic cell score (SCS, p = 0.032). The CSN3 (HQ677596: c.536C>T) was positively associated with SCS (p = 0.005) and milk urea (p = 0.04). Favorable effects on daily milk yield (dMY, p = 0.028), fat (dFP, p = 0.027) and protein (dPP, p = 0.050) percentages were observed for the LPL. Conversely, the SCD did not show any association with milk traits. This is the first example of a confirmation study carried out in the Mediterranean river buffalo for genes of economic interest in the dairy field, and it represents a very important indication for the preselection of young bulls destined for breeding programs aimed at more sustainable dairy production.

Polymorphism at the CSN1S1 Locus and Energy Intake Level Affect Milk Traits and Casein Profiles in Rossa Mediterranea Goats.

A total of twenty-seven Rossa Mediterranea lactating goats, consisting of nine homozygous for strong alleles (AA), twelve heterozygous (AF) and six homozygous for weak alleles (FF) at the CSN1S1 locus, were used to evaluate the effect of genotype, diet and genotype × diet interaction on goat milk traits and casein profile. The goats were used in a 3 × 3 factorial arrangement of treatments, with three genotypes (AA, AF and FF) and three different energy intake levels: high (H), medium (M) and low (L). The diets supplied a complete pelleted feed containing 65% of alfalfa hay, respectively, at 150%, 100% and 70% of the total energy requirements. Milk yield was significantly affected by the genotype and diet: Lower levels were found in FF goats than in AA and AF genotypes (673.7 vs. 934.5 and 879.8 d/g, respectively; p = 0.002) as well as in goats fed with the L diet (651.5 vs. 1041 and 852.9 g/d for H and M diet, respectively, p < 0.001). The genotype influenced the casein profile. Specifically, AA goat milk exhibited higher concentrations of total casein and αs1-casein compared to AF and FF genotypes (for total casein and αs1-casein, respectively: 24.9 vs. 20.4 and 19.8 g/kg, p = 0.001; 7.2 vs. 3.7 and 0.7 g/kg, p < 0.001), while the FF genotype showed higher values for αs2-casein concentrations compared to homozygous AA and heterozygous AF goats (3.1 vs. 2.4 and 2.5 g/kg, respectively, p < 0.001). A significant genotype x diet interaction occurred for αs2-casein levels (g/kg) (p = 0.034) and αs1-casein yields (p = 0.027): The αs2-casein level was not affected by the diet in AA goats, whereas it increased with energy intake in AF and FF genotypes. Conversely, the αs1-casein yield gradually increased with energy intake in AA and AF groups, whereas the diet in FF goats did not modify it. The results demonstrated that high energy input, as well as the strong allele at the CSN1S1 locus, enhanced milk production and casein concentrations. Furthermore, they confirmed the existence of an interaction between αs1-casein polymorphism and diets, influencing the milk casein composition and yield.

A Genotyping Method for Detecting Foreign Buffalo Material in Mozzarella di Bufala Campana Cheese Using Allele-Specific- and Single-Tube Heminested-Polymerase Chain Reaction.

Mozzarella di Bufala Campana (MdBC) cheese is a Protected Designation of Origin (PDO) product that is important for the economy and cultural heritage of the Campania region. Food fraud can undermine consumers' trust in this dairy product and harm the livelihood of local producers. The current methods for detecting adulteration in MdBC cheese due to the use of buffalo material from foreign countries could exhibit limitations associated with the required use of expensive equipment, time-consuming procedures, and specialized personnel. To address these limits here, we propose a rapid, reliable, and cost-effective genotyping method that can detect foreign buffalo milk in a counterpart from the PDO area and in MdBC cheese, ensuring the quality and authenticity of the latter dairy product. This method is based on dedicated allele-specific and single-tube heminested polymerase chain reaction procedures. By using allele-specific primers that are designed to detect the nucleotide g.472G>C mutation of the CSN1S1Bbt allele, we distinguished an amplicon of 330 bp in the amplification product of DNA when extracted from milk and cheese, which is specific to the material originating from foreign countries. By spiking foreign milk samples with known amounts of the counterpart from the PDO area, the sensitivity of this assay was determined to be 0.01% v/v foreign to PDO milk. Based on a rough estimate of its simplicity, reliability, and cost, this method could be a valuable tool for identifying adulterated buffalo PDO dairy products.

Variations in Casein Genes Are Associated with Milk Protein and Fat Contents in Sarda Goats (Capra hircus), with an Important Role of CSN1S2 for Milk Yield.

This work aimed to assess the variability of casein genes in a population of 153 bucks and 825 lactating does of the Sarda breed, and to perform association analysis between polymorphic sites and milk yield and composition traits. To genotype the casein genes, we chose an SNP panel including 44 SNPs mapping to the four casein genes CSN1S1, CSN2, CSN1S2, and CSN3. Genotyping (made by KASP™ genotyping assay, based on competitive allele-specific PCR) revealed the high variability of the Sarda goat, and haplotype analysis revealed linkage disequilibrium (LD) between CSN1S1 and CSN2 genes, in addition to two LD blocks within the CSN1S2 and two LD blocks within the CSN3 gene, in bucks and does. Association analysis revealed that variability at all four casein genes was associated with milk protein content, total solids, and milk energy. The three Ca-sensitive casein genes were associated with lipid content, and CSN1S2 showed a unique pattern, with intron variants associated with milk yield, in addition to milk pH, NaCl, and SCS (Somatic Cell Score). This information might prove useful in selection schemes and in future investigations aiming to better understand the biology of lactation, and the direct link between genotype and phenotype.

Mutation of Signal Transducer and Activator of Transcription 5 (STAT5) Binding Sites Decreases Milk Allergen αS1-Casein Content in Goat Mammary Epithelial Cells.

αS1-Casein (encoded by the CSN1S1 gene) is associated with food allergy more than other milk protein components. Milk allergy caused by αS1-casein is derived from cow milk, goat milk and other ruminant milk. However, little is known about the transcription regulation of αS1-casein synthesis in dairy goats. This study aimed to investigate the regulatory roles of signal transducer and activator of transcription 5 (STAT5) on αS1-casein in goat mammary epithelial cells (GMEC). Deletion analysis showed that the core promoter region of CSN1S1 was located at -110 to -18 bp upstream of transcription start site, which contained two putative STAT5 binding sites (gamma-interferon activation site, GAS). Overexpression of STAT5a gene upregulated the mRNA level and the promoter activity of the CSN1S1 gene, and STAT5 inhibitor decreased phosphorylated STAT5 in the nucleus and CSN1S1 transcription activity. Further, GAS site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays revealed that GAS1 and GAS2 sites in the CSN1S1 promoter core region were binding sites of STAT5. Taken together, STAT5 directly regulates CSN1S1 transcription by GAS1 and GAS2 sites in GMEC, and the mutation of STAT5 binding sites could downregulate CSN1S1 expression and decrease αS1-casein synthesis, which provide the novel strategy for reducing the allergic potential of goat milk and improving milk quality in ruminants.

Genetic Diversity in Casein Gene Cluster in a Dromedary Camel (C. dromedarius) Population from the United Arab Emirates.

Genetic polymorphisms, causing variation in casein genes (CSN1S1, CSN1S2, CSN2, and CSN3), have been extensively studied in goats and cows, but there are only few studies reported in camels. Therefore, we aimed to identify alleles with functional roles in the United Arab Emirates dromedary camel (Camelus dromedarius) population to complement previous studies conducted on the same species. Using targeted next-generation sequencing, we sequenced all genes in the casein gene cluster in 93 female camels to identify and characterize novel gene variants. Most variants were found in noncoding introns and upstream sequences, but a few variants showed the possibility of functional impact. CSN2 was found to be most polymorphic, with total 91 different variants, followed by CSN1S1, CSN3 and CSN1S2. CSN1S1, CSN1S2 and CSN2 each had at least two variants while CSN3 had only one functional allele. In future research, the functional impact of these variants should be investigated further.

Transcripts and protein levels of CSN1S1 and CSN3 genes in dairy cattle mammary gland secretory tissue during chronic staphylococcal infection.

Our objective was to determine the influence of chronic coagulase-positive staphylococci (CoPS) or coagulase-negative staphylococci (CoNS) infection on the mRNA and protein levels of two main milk proteins responsible for cheese curd quantity and quality, alpha-S1-casein (CSN1S1) and kappa-casein (CSN3). Measurements were made in cow mammary parenchyma with a prevalence of secretory tissue (MGST). Samples of MGST were collected from the separate quarters and divided into CoPS, CoNS and bacteria-free (H) groups according to the microbiological status of the quarter milk. No differences in CSN1S1 and CSN3 mRNA level were found between groups, however, CSN1S1 protein level was significantly higher in the H group than the CoNS group, and CSN3 protein level was significantly higher in H than CoPS group. Hence, while the CSN1S1 and CSN3 genes appear to be constitutively expressed at the mRNA level in dairy cow MGST during mastitis, CoNS infection negatively affected CSN1S1 protein level, and CoPS infection negatively affected CSN3 protein level. The lack of change at the mRNA level suggests that staphylococcal infection may affect the post-transcriptional or post-translational modifications.

Validation of CSN1S1 transcriptional expression, promoter methylation, and prognostic power in breast cancer using independent datasets.

Breast cancer ranked second among most frequent cancer in the world playing a significant role in mortality rate. Having prior knowledge on differentially expressed genes in breast cell carcinoma elucidated important indications to understand the molecular mechanism underneath breast carcinogenesis. In this study we have investigated the distinguished CSN1S1 expression in human breast cancer. We have analyzed CSN1S1 mRNA expression between cancer and normal tissues using TCGA datasets. Moreover, analysis including promoter methylation, mutations, prognosis, co-expression, gene ontology, and pathways of CSN1S1 were performed by the TCGA Wanderer, UCSC Xena, cBioPortal, PrognoScan, UALCAN, and Enricher server. We have observed low mRNA expression and high promoter methylation of CSN1S1 in cancer tissues compared to normal tissues. Furthermore, we have also identified low mRNA expression in clinicopathological patients, as well as 9 deleterious mutations with highly co-expressed protein MRC1, and significantly related signaling pathways. We have found a positive correlation between the lower expression of CSN1S1 and patients surviving with breast cancer. Here we have concluded that CSN1S1 acts as a biomarker for the surveillance and prognosis of breast cancer, and also works as a novel therapeutic target at the molecular and pathway levels.

Influence of the Casein Composite Genotype on Milk Quality and Coagulation Properties in the Endangered Agerolese Cattle Breed.

The aim of this study was the characterization of CSN1S1, CSN2 and CSN3 genetic variability in Agerolese cattle, and the investigation of the effect of casein composite genotypes (CSN1S1, CSN2 and CSN3) on quality and coagulation traits of the corresponding milk. To these purposes, blood and milk from 84 cows were sampled and analysed. Allele frequencies at CSN2 and CSN3 revealed no Hardy-Weinberg equilibrium in the population with a prevalence of allele A2 for CSN2 and allele B for CSN3. BBA1A2AB and BBA2A2AB composite genotypes were the most common in the population. BBA1A2AB showed a higher total solids and fat content (12.70 ± 0.16 and 3.93 ± 0.10, respectively), while BBA2A2BB showed the best coagulation properties (RCT 12.62 ± 0.81; k20 5.84 ± 0.37; a30 23.72 ± 1.10). Interestingly, the A2 allele of CSN2 was very widespread in the population; thus, it will be intriguing to verify if A2A2 Agerolese cattle milk and the derived cheese may have better nutraceutical characteristics.

Oxidative Status of Goats with Different CSN1S1 Genotypes Fed ad Libitum with Fresh and Dry Forages.

Forty late-lactation Girgentana goats were used to study the effect of diets fed ad libitum and αS1-casein (CSN1S1) genotype on redox balance. The goats genotyped at CSN1S1 locus (A/A, A/F) were subjected to four feeding treatments different for percentage inclusion of dry and fresh forage: DAF100 (98% of Dry Alfalfa Forage), DAF65 (65% of Dry Alfalfa Forage), FSF100 (100% of Fresh Sulla Forage) and FSF65 (65% of Fresh Sulla Forage). Blood samples were analyzed for superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity, reactive oxygen metabolites (ROMs), biological antioxidant potential (BAP) and non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), albumin, glucose and cholesterol contents. The oxidative stress index (OSI) was calculated as percentage ratio of ROMs to BAP. Redox balance was improved by Sulla inclusion, as reflected in the lower OSI values found in FSF100 and FSF65 groups. DAF100 group displayed the highest GPX activity, while other groups exhibited the highest SOD activity. Fresh forage diets increased albumin concentration while no effect of tested factors was noted on glucose, NEFA, BHBA and cholesterol contents. The interaction diet × genotype was significant only for GPX activity. GPX and albumin were negatively correlated and were correlated positively and negatively with ROMs, respectively. Diet rather than genotype affects redox balance in dairy goats and a possible role of forage polyphenol compounds on oxidative status needs to be tested in future studies.

Lama glama αS1-casein: Identification of new polymorphisms in the CSN1S1 gene.

South American camelids have been poorly genetically investigated and little information is available in llamas (Lama glama) regarding the diversity of the caseins at the protein and gene level. Exon skipping and duplication events previously reported in the αS1-casein gene (CSN1S1) led us to investigate the genetic variability at this locus. Seventy-two positive clones for the αS1-casein transcripts were analyzed and randomly sequenced. The comparative analysis of the sequences revealed 2 transitions, c.366A>G and c.690T>C, at the 10th nucleotide of exon 12 and 94 bp of exon 19, respectively. These SNP are responsible for 2 amino acid changes, Ile→Val in position 86 and Tyr→His in position 194 of the mature protein. Both polymorphisms clarify the genetic events behind the protein variants A and B. This result was confirmed by isoelectric focusing analysis of llama milk samples. Quick methods based on PCR-RFLP and allele-specific PCR were set up for allelic discrimination in a population of 128 animals. Based on genotyping results, 4 haplotypes were observed and the estimated frequencies indicated B as the most common haplotype (0.629) in the investigated population. These data add knowledge to the genetic variability of a species little investigated, and open opportunity for new investigation in the field of milk protein for South American camelids, including the possibility, in the future, to select alleles with favorable characteristics.

Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants.

Comparison of milk oligosaccharides between goats with and without the genetic ability to synthesize αs1-casein.

Milk oligosaccharides (OS)-free complex carbohydrates-confer unique health benefits to the nursing neonate. Though human digestive enzymes cannot degrade these sugars, they provide nourishment to specific commensal microbes and act as decoys to prevent the adhesion of pathogenic micro-organisms to gastrointestinal cells. At present, the limited quantities of human milk oligosaccharides (HMO) impede research on these molecules and their potential applications in functional food formulations. Considerable progress has been made in the study of OS structures; however, the synthetic pathways leading to their synthesis in the mammary gland are poorly understood. Recent studies show that complex OS with fucose and N-acetyl neuraminic acid (key structural elements of HMO bioactivity) exist in goat milk. Polymorphisms in the CSN1S1 locus, which is responsible for synthesis of αs1-casein, affect lipid and casein micelle structure in goat milk. The present study sought to determine whether CSN1S1 polymorphisms also influence goat milk oligosaccharide (GMO) production and secretion. The GMO compositions of thirty-two goat milk samples, half of which were from genotype A/A (αs1-casein producers) and half from genotype O/O (αs1-casein non-producers), were determined with nanoflow liquid chromatography high-accuracy mass spectrometry. This study represents the most exhaustive characterization of GMO to date. A systematic and comprehensive GMO library was created, consolidating information available in the literature with the new findings. Nearly 30 GMO, 11 of which were novel, were confirmed via tandem mass spectrometric analyses. Six fucosylated OS were identified; 4 of these matched HMO compositions and three were identified for the first time in goat milk. Importantly, multivariate statistical analysis demonstrated that the OS profiles of the A/A and O/O genotype milks could be discriminated by the fucosylated OS. Quantitative analysis revealed that the goat milk samples contained 1.17 g/L of OS; however, their concentration in milks from A/A and O/O genotypes was not different. This study provides evidence of a genetic influence on specific OS biosynthesis but not total OS production. The presence of fucosylated GMO suggests that goat milk represents a potential source of bioactive milk OS suitable as a functional food ingredient.

Estudo do alelo g do gene da alphas1-caseína em uma população de cabras leiteiras / Estudio del alelo g del gen de la alphas1- caseína en una población de cabras lecheras / Study on the g allele of the alphas1-casein gene in dairy goats

Devido ao diminuído potencial alergênico do leite de cabra, em relação ao leite de vaca, as proteínas do primeiro têm sido investigadas e mostram um amplo polimorfismos da ?S1-caseína, variando de produção nula até 7 g/L. No lócus gênico da ?S1-caseína já foram encontrados 18 alelos, sendo o alelo G relacionado a baixos níveis de ?s1-caseína (0,6 g/L), decorrente de uma transição G?A (guanina por adenina) no sítio doador de splice do intron 4, levando à perda do exon 4 durante o processamento do RNA mensageiro. No Brasil existem poucos relatos de estudos sobre o polimorfismo genético desta proteína. Assim, este trabalho teve como objetivo estabelecer um protocolo para detecção do alelo G do gene da ?S1- caseína e verificar sua existência em um grupo de animais caprinos. Para isto, leucócitos foram utilizados para isolamento de DNA com o reagente CTAB e um par de primer, desenhado com base na seqüência do GenBank (AJ504710), possibilitou a amplificação da região gênica de interesse, gerando produtos de PCR com tamanho de 550 pb. Após a digestão com a enzima AciI, foram observados dois fragmentos com tamanhos de 308 e 242 pb, indicando a presença da guanina no sítio doador de splice. Alguns animais, suspeitos de portarem o alelo G, tiveram amostras de DNA seqüenciadas. Os resultados mostraram inexistência da transição de base e, conseqüentemente, a inexistência do alelo G nos 80 animais estudados, indicando a necessidade de investigação de um número maior de animais, por se tratar de um alelo raro